This invention relates to systems for expansion of cell populations.
The origin of all the cells in blood and in the immune system is the hematopoietic stem cell (HSC). Each HSC has the potential to differentiate into at least eight separate blood cell lineages within the myeloid and lymphoid blood cell compartments. It has been estimated through successive generational analysis that one HSC has the potential to produce up to fifty million differentiated progeny. See U.S. Pat. No. 5,061,620, the disclosure of which is incorporated herein by reference in its entirety.
This enormous potential could be exploited if, starting from a small number of HSCs, a large pool of HSCs could be grown in culture without significant differentiation during expansion. This pool of HSCs could then be used to restore or supplement an immune system and/or blood forming system compromised by, e.g., radiation or chemotherapy. The pool would also be a valuable tool in the design, development and testing of diagnostic and therapeutic agents used in the treatment of immune system and/or blood forming disorders.
Efforts have been made to develop a system that would grow HSCs ex vivo and control cell proliferation and differentiation. Typically, these efforts have involved batch culture of a mixed population of cells that have been initially separated from a relatively large volume of blood.
The invention is based on our discovery that a predetermined target population of cells, in particular renewable cells, e.g., relatively undifferentiated cells including HSCs, can be clonogenically expanded in a system that either (a) positively selects for cells of the target population, or (b) negatively selects out non-target cells. Selection of this type occurs concurrently with cell growth or intermittently during cell growth. Advantageously, by selectively controlling the relative populations of cells in the system, the invention allows greater expansion of the target population. This selective population control reduces feedback inhibitions, influences factor and substrate consumption rates, and minimizes other limiting factors that tend to occur in conventional batch cultures.
In one aspect, the invention features a method of selective expansion of a predetermined target population of cells that includes: (a) introducing a starting sample of cells into a growth medium; (b) causing cells of said predetermined target cell population to divide; and (c) contacting the cells in the growth medium with a selection element, comprising a plurality of selective binding molecules with specific affinity for a predetermined population of cells, so as to select cells of said predetermined target population from other cells in the growth medium. The selection element may use positive selection (the selective binding molecules are specific for target cells), or negative selection (the selective binding molecules are specific for non-target cells). The method may also include contacting the starting cells with a reverse selection element employing the opposite type of selection.
In preferred embodiments, the starting sample of cells includes target cells, and the expansion is clonogenic. Alternatively, the starting sample of cells includes progenitors of said target cells. In some preferred embodiments, the selection element comprises a solid support to which said selective binding molecules are bound. The growth medium can be disposed in or caused to flow through a chamber. The growth medium may also be caused to recycle through the chamber, flowing from an inlet, through the chamber, to an outlet of the chamber, and returning from the outlet to the inlet via a conduit. It is further preferred that the oxygen saturation of the growth medium be regulated to be from 0% to 20% relative to the solubility of oxygen in said fluid at equilibrium with air at 37xc2x0 C. and 1 atm pressure.
The invention also features a method of selective expansion of a predetermined target population of cells including: (a) introducing fluid containing a plurality of cells into a growth medium; (b) causing cells of said predetermined target cell population to divide; and (c) selecting cells of said predetermined target population from other cells in the growth medium; wherein steps (b) and (c) are carried out substantially simultaneously.
The invention also features a system for continuous selective expansion of a predetermined target population of cells. The system includes (a) a growth medium for supporting cell division; (b) a chamber for receiving said growth medium; and (c) a selection element, positioned to contact said growth medium during or after cell division. The selection element includes a plurality of binding sites bearing a selective binding molecule. The selective binding molecule can have (i) a specific affinity for cells of said predetermined target cell population or (ii) a specific affinity for non-target cells and substantially less affinity for target cells. If desired, the system can further include a reverse selection element having the opposite type of affinity.
One system of the invention for continuous selective clonogenic expansion of relatively undifferentiated cells includes: (a) a tube containing a plurality of beads of a size which permits a plurality of the undifferentiated cells to grow thereon, the beads bearing on their surfaces a plurality of selective binding molecules capable of binding to a surface antigen present on the relatively undifferentiated cells, wherein the surface antigen is not present on relatively differentiated cells; (b) means for continuously providing nutrients to the relatively undifferentiated cells growing on the beads, wherein the nutrients are delivered via a fluid which flows through the tube and past the beads so that the relatively undifferentiated cells in the tube divide and at least a portion of relatively undifferentiated cells exit the tube with the fluid; and (c) means for continuously harvesting the portion of the relatively undifferentiated cells that exit the tube.
The invention can be used to provide stem cells (HSCs) useful for enhancing the immune system of a patient. The patient""s blood or bone marrow is withdrawn (or an allogeneic stem-cell containing sample is provided); stem cells are expanded and harvested according to the invention; and then those cells are re-introduced into the patient, where they will facilitate enhancement or reconstitution of the patient""s immune and/or blood forming system.
Preferably, the sample taken from the patient is relatively small, e.g., less than about 100 to 200 ml, to minimize trauma to the patient. The preferred potency and dosage of the undifferentiated cells to administer to the patient, and duration of administration, will vary depending upon the condition of the patient""s immune or blood forming system, but would generally be expected to be in the range of from about 100 to 1xc3x97106 cells/kg body wgt/dose/day.
Alternatively, the invention can be used to provide to a patient a predetermined population of relatively differentiated cells, by providing a sample containing a population of cells which cells are the progenitor to the predetermined population, and using the system of the invention to cause the progenitor cells to proliferate and differentiate to form the predetermined population of cells, e.g., by providing the cells with a growth factor which will cause differentiation. For example, the differentiated cells may be lymphoid precursors, myeloid precursors or erythroid precursors. The invention can also be used to provide to a patient a therapeutic compound produced by a population of cells by using the system of the invention to proliferate cells of the population and to cause the population to produce the substance.
The term xe2x80x9ccontinuous,xe2x80x9d as used herein, refers to a process which proceeds substantially constantly, with dividing cells being removed from the system shortly after they are born, rather than remaining in culture as in a conventional batch process. This term, as used herein, does not imply that the process is necessarily a steady state process, although in some preferred embodiments steady state may potentially be reached.
The term xe2x80x9cspecific affinity,xe2x80x9d as used herein, refers to a tendency to bind a surface molecule or feature that is present on a distinct population of cells and absent on cells not of the population. Examples of such surface molecules or features include but are not limited to cell adhesion molecules, antigens, carbohydrates and functional or non-functional receptors.
The term xe2x80x9cnon-specific interaction,xe2x80x9d as used herein, refers to interactions which interfere with and/or reduce the efficiency of desired specific interactions.
The invention provides tremendous potential for continuous long-term production of cell populations which can be supplied to a patient or other user of the cells almost as soon as the cells are born (or frozen as soon as they are harvested and supplied in frozen form at any desired time). The system can be used as a research tool for studying the effects of biopharmacological agents, growth factors, mitogens and the like, and also as a diagnostic tool, e.g., to gauge the hematopoietic potential of a patient.
The invention can be used not only to proliferate relatively undifferentiated cells, but also to produce populations of other cells simply by selecting the appropriate growth factor to supply to the system during expansion, and to produce desired cell by-products, e.g., those which could be administered as therapeutic compounds to a patient. Because the initial cell sample can be autologous, the cell populations or cell by-products produced are likely to be readily accepted by the patient from whom the cell sample was obtained.
Because the contents of the system can be frozen, a sample can be taken from a patient, introduced into the system, and then saved for a prolonged period for later use when needed, e.g., when the patient""s immune system or blood forming system is challenged.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.